Fishborne Zoonotic Intestinal Trematodes, Vietnam Do Trung Dung,*† Nguyen Van De,‡ Jitra Waikagul,† Anders Dalsgaard,§ Jong-Yil Chai,¶ Woon-Mok Sohn,# and K

RESEARCH

Fishborne Zoonotic Intestinal Trematodes, Vietnam Do Trung Dung,*† Nguyen Van De,‡ Jitra Waikagul,† Anders Dalsgaard,§ Jong-Yil Chai,¶ Woon-Mok Sohn,# and K. Darwin Murrell§

Although fi shborne zoonotic trematodes that infect the 10–20 years, a second large group of FZTs, the so-called liver are well documented in Vietnam, intestinal fi shborne minute intestinal fl ukes, has been increasingly recognized zoonotic trematodes are unreported. Recent discoveries of as widely distributed and a cause of illness (1–8). the metacercarial stage of these fl ukes in wild and farmed The exponential increase in aquaculture is suggested fi sh prompted an assessment of their risk to a community to be the major cause of the emergence of FZTs in east that eats raw fi sh. A fecal survey of 615 persons showed a and Southeast Asia (2,3). For example, in the People’s Re- trematode egg prevalence of 64.9%. Infected persons were treated to expel liver and intestinal parasites for specifi c public of China, the land devoted to aquaculture increased identifi cation. The liver trematode Clonorchis sinensis was 75% (to 4.9 million hectares) since 1970, accompanied by recovered from 51.5%, but >1 of 4 intestinal species of the a tripling of cases of infection with C. sinensis (3). The family Heterophyidae was recovered from 100%. The most association of O. viverrini in Thailand and Lao People’s numerous were Haplorchis spp. (90.4% of all worms recov- Democratic Republic with fi sheries has also been reported ered). These results demonstrate that fi shborne intestinal (9,10). However, wild fi sh are also frequently infected, but parasites are an unrecognized food safety risk in a country epidemiologic information to compare relative infection whose people have a strong tradition of eating raw fi sh. risks from eating wild fi sh and farmed fi sh in many FZT- endemic loci is insuffi cient (1,4). oodborne parasites are widespread and more common A recent review of publications on FZTs in Vietnam Fthan generally recognized. Among these parasites, fi sh- indicated infections with only liver fl ukes (C. sinensis and borne zoonotic trematodes (FZTs) are estimated to infect O. viverrini) in humans (11). However, recent Vietnamese >18 million persons; worldwide the number at risk may be surveys for zoonotic parasites in cultured and wild fi sh in much greater (1–3). The FZTs include many species, espe- northern and southern Vietnam identifi ed metacercarial cially representatives of the families Heterophyidae, Echi- stages of several zoonotic intestinal trematode species in nostomatidae, and Opisthorchiidae. Although their meta- fi sh (12,13). This fi nding is of concern because fi sh produc- cercarial cysts are easily inactivated by heating at 60°C tion has increased 9.3-fold (to 400,000 tons) over the past or freezing to –20°C, they are highly prevalent in many 40 years in Vietnam (3). Furthermore, human intestinal regions, especially in Asia where food traditions include fl ukes are highly prevalent in neighboring countries such as eating raw or improperly cooked fi sh dishes (Figure 1) (4). Thailand (14), Lao People’s Democratic Republic (15), and The fi shborne liver fl ukes Clonorchis sinensis, Opisthorchis the People’s Republic of China (16), which further raises viverrini, and O. felineus cause cholangitis, pancreatitis, the issue of whether human infections might be present in and cholangiocarcinoma in humans (4–6). During the past Vietnam but overlooked because of diagnostic diffi culties in differentiating liver and intestinal fl uke eggs in fecal ex- *National Institute of Malariology, Parasitology and Entomology, aminations (11,17). A more reliable approach to detect and Hanoi, Vietnam; †Mahidol University, Bangkok, Thailand; ‡Hanoi characterize human FZTs is to treat egg-positive patients Medical University, Hanoi, Vietnam; §University of Copenhagen, and recover and identify the expelled adult worms (7,15). Frederiksberg, Denmark; ¶Seoul National University College of We conducted a study in April 2005 in Nam Dinh Medicine, Seoul, South Korea; and #Gyeonsang National Univer- Province, an area of Vietnam in which persons are known sity, Jinju, South Korea for eating raw fi sh, and where previous investigations have

1828 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 Fishborne Zoonotic Intestinal Trematodes, Vietnam

NIMPE by the World Health Organization and originally obtained from Vestegaard Frandsen Pvt. Ltd. (New Delhi, India). Fecal slides were examined by light microscopy (×400). Helminth eggs were identiﬁ ed and enumerated, and the number of eggs was multiplied by 23 to obtain the number of eggs per gram (epg) of feces.

Parasite Expulsion Thirty-three persons who had >1,000 epg on fecal examinations were selected for worm expulsion. Selected patients were asked to eat a light liquid dinner the evening before treatment. The following morning they were given oral praziquantel, 25 mg/kg; 1 hour later, they were given

a saturated solution of 30 g of MgSO4 dissolved in water. Subsequently, 3–4 consecutive posttreatment stools were collected. Worms were recovered by a series of washing steps (15). Figure 1. Typical dish of raw ﬁ sh (slices of silver carp) sold in Vietnamese restaurants.

shown a high prevalence of liver fl ukes (11,13). Identifi cation of worms expelled from egg-positive persons showed that intestinal FZTs are present in Vietnam and represent a major public health risk for a population with the habit of eating raw fi sh.

Materials and Methods

Site, Sampling, and Examination Procedure A cross-sectional survey for fecal eggs was conducted in 2 communes in Nghia Hung District, Nam Dinh Prov- ince, Vietnam, southeast of the capital of Hanoi (Figure 2), a clonorchiasis-endemic area (11). The rural population in these communes is mostly farmers with fi sh ponds that are integrated into their farming systems, e.g., pig farming. In 2005, the Nghia Phu commune had a population of 9,608, including 2,214 families, and the Nghia Lac commune had a population of 9,147, including 2,160 families. House- holds in these 2 communes were randomly selected from a list provided by community authorities, and from each household 1 man or 1 woman who was head of household was selected. Trained personnel delivered labeled plastic containers to the selected persons and instructed them how to collect a fecal sample and store it until it was retrieved the next day. The label requested the person’s name, age, address, and date of stool collection. Permission to conduct this research was obtained from the National Institute of Malariology, Parasitology and Entomology (NIMPE), Hanoi, and the Faculty of Tropical Medicine, Mahidol University, Bangkok. Each study par- ticipant signed a consent form, which is on fi le at NIMPE. Figure 2. Map of Vietnam showing location of Nam Dinh Province, From each stool sample, 2 Kato-Katz smears were pre- investigated for fi shborne zoonotic trematode infections, April pared and analyzed by using the standard kit provided to 2005.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1829 RESEARCH

All persons who were positive for eggs were provided Table 1. Helminth infections in persons living in Nghia Phu and free drug treatment. Patients with nematode infections were Nghia Lac communes, Nam Dinh Province, Vietnam, April 2005 given 1 dose of albendazole (400 mg) or mebendazole (500 Fecal examination result, mg), and patients with trematode infections were given pra- Helminth egg species or type no. positive (%) Small trematodes (<50 μm long) 399 (64.9) ziquantel (25 mg/kg, 3× a day for 1 day). Large trematodes (>50 μm long) 5 (0.8) Ascaris lumbricoides 243 (39.5) Identifi cation of Adult Worms Recovered Trichuris trichiura 358 (58.2) Adult worms were identifi ed by direct light micros- Hookworm 19 (3.1) copy while still alive; those that needed further examina- Total positive 554/615 (90.1) tion were fi xed in 10% formalin and stained with Semi- chon acetocarmine, mounted on a slide, and measured (18). cantly between age groups; it was signifi cantly higher for Identifi cations were made by using published taxonomic those >40 years of age (χ2 7.95, p<0.05). In contrast, wom- references (19,20). en did not show a signifi cant difference in infection rates between age groups (χ2 0.85, p>0.05). Data Analysis Most persons with small trematode eggs showed low Results of fecal examinations for helminth eggs were infection intensity (epg); 344 (86.2%) of 399 shed <1,000 analyzed for prevalence and intensity of infection (epg), as epg, and 55 (13.8%) of 399 shed 1,000–9,999 epg. Infection measured by enumeration of eggs per gram of feces. Spe- intensity differed signifi cantly between those <40 years of cies infection rates (number of expelled worms) were de- age and those >40 years of age (χ2 4.17, p<0.05) (Table 2). scriptively analyzed by using SPSS version 11.0 software The prevalence of A. lumbricoides was 39.3% in men (SPSS Inc., Chicago, IL, USA) and χ2 and Student t tests. and 42.3% in women. There was a signifi cant increase in prevalence with age only in women (χ2 6.4, p<0.05). Infec- Results tion with T. trichiura infection did not differ signifi cantly by sex or age (p>0.05). Parasite Diversity, Prevalence, and Intensity A total of 615 persons, 563 men (91.5%) and 52 women FZT Species Identifi cation (8.5%), were selected and submitted stool for examination. Trematodes responsible for releasing small eggs Fecal egg examinations showed that 554 persons (90.1%) were identifi ed by using morphologic characterization of were positive for helminth parasites (Table 1). Trichuris adult stages expelled from patients. A total 15,185 adult trichiura (whipworm) nematode eggs were found in 58.2% worms were collected from 33 patients. The number and of the stool samples. A total of 64.9% were infected with prevalence of individual species of expelled trematodes are small trematode eggs (<50 μm long), and 39.5% were shown in Table 3. C. sinensis and 4 species of intestinal infected with Ascaris lumbricoides (roundworm). Hook- fi shborne zoonotic fl ukes were identifi ed (Figure 3); C. si- worm eggs (3.1%) and large (>50 μm long) trematode eggs nensis was isolated from 51.5% of patients. Intestinal fl uke (0.8%) were infrequently seen (Table 1). Multiparasitism species identifi ed (mean body length × width measurements was common in this community, with 65.1% of the persons in μm) were Haplorchis pumilio (632 × 291), H. taichui expelling eggs having >2 species or types of eggs. Small (756 × 421), H. yokogawai (760 × 400), and Stellantchas- trematode eggs, all <50 μm long, were presumed to be ei- mus falcatus (468 × 298). Prevalence of intestinal fl ukes ther those of C. sinensis or of intestinal trematodes of the was H. pumilio, 100%; H. taichui, 69.7%; H. yokogawai, family Heterophyidae. However, differentiation was not 6.1%; and S. falcatus, 3.0%. H. pumilio was the most com- considered reliable by light microscopy, and selected per- mon trematode (90.4%) of all worms isolated; 13,734 adult sons were treated to expel their helminth parasites. worms were isolated from 33 persons (mean 416.2); 1 pa- On the basis of egg count data, small trematode infec- tient expelled 4,525 worms. The plantborne intestinal pig tion prevalence differed signifi cantly between men (68.7%) trematode Fasciolopsis buski was isolated from 1 patient. and women (23.1%) (χ2 43.56, p<0.05). The infection rate Multiple infections with FZTs were common (Figure for small trematode infection in men also differed signifi - 4): 54.5% of patients were infected with 2 trematode spe-

Table 2. Intensity of small trematode infections in 2 age groups, Nam Dinh Province, Vietnam, April 2005* Age group No. positive No. (%) with light infection† No. (%) with moderate infection† <40 y 111 102 9 >40 y 288 242 46 Total 399 344 (86.2) 55 (13.8) *epg, eggs per gram (of feces). †Light infection = 1–999 epg; moderate infection = 1,000–9,999 epg.

1830 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 Fishborne Zoonotic Intestinal Trematodes, Vietnam

Table 3. Species and no. trematode adult worms recovered, Vietnam, Nam Dinh Province, April 2005 Trematode sp. No. positive persons (%) Total worms recovered Average no. worms recovered (range) Liver fluke Clonorchis sinensis 17 (51.5) 72 4.2 (1–18) Intestinal flukes Haplorchis pumilio 33 (100) 13,734 416.2 (1–4,525) H. taichui 23 (69.7) 1,323 40.1 (1–307) H. yokogawai 1 (3.0) 3 3 Stellantchasmus falcatus 2 (6.1) 52 26 (15–37) Fasciolopsis buski 1 (3.0) 1 1 cies, 33.3% with 3 species. A total of 9% were infected Intensifi cation of aquaculture, use of human and animal with only 1 species. One person (3%) was infected with 4 manure for pond fertilization, and increased consumption FZT species and F. buski. of fi sh because of increasing affl uence by a population with a tradition of eating raw fi sh may be contributing factors Discussion for infection. These issues need to be investigated if effec- Our results demonstrate that zoonotic fi shborne intes- tive means for prevention of transmission are to be devel- tinal trematodes are endemic in Vietnam. These trematodes oped. Use of manure and waste water in aquaculture is a represent, to our knowledge, a new and previously unrecog- well-recognized risk factor for trematode infections in fi sh nized public health problem. To our knowledge, in the many (2,12,13) and has been the focus of 2 hazard analysis, criti- publications on human parasites originating in Vietnam cal control point–based control projects (4). Major sources since the 19th century colonial era, no reports on these intes- of infected fi sh responsible for trematode transmission to tinal fi shborne parasites have appeared (11). Whether this humans must be ascertained because FZT metacercariae zoonosis is newly emerging in Vietnam because of changes have been found in both wild and farmed fi sh in Vietnam, in agriculture/aquaculture, demographics, social, or environ- as well as elsewhere in Asia (4,10,12–16). mental changes or if it has been overlooked because of diag- Public health and agricultural/fi shery agencies should nostic problems is not known. However, snail vectors (e.g., consider intestinal and liver fl ukes as an FZT complex be- Melanoides tuberculata) and suitable vertebrate intermedi- cause they share most biologic features and are risk factors ate (fi sh) and reservoir hosts (fi sh-eating birds, dogs, cats, for human infection. Although intestinal fl ukes are less well pigs) for FZTs are common in this country (1,4,11,19,21). characterized clinically than liver fl ukes, they are increas- Furthermore, H. taichui, H. pumilio, H. yokogawai, and S. ingly being recognized as a cause of intestine, heart, brain, falcatus are endemic in neighboring countries such as Thai- and spinal cord abnormalities in humans (1,4,8,23). land (14), Lao People’s Democratic Republic (10,15), and The potential economic effect of FZTs on alleviation the People’s Republic of China (16). of poverty is also a concern. Aquaculture in Vietnam is a It is puzzling why zoonotic heterophyids have only re- major economic activity in rural areas. During 2000–2006, cently been isolated from fi sh in Vietnam (12,13,22) if they Vietnam tripled the value of its export of fi sh, increasing its are endemic. These parasites may have been recently intro- revenue to >3 billion US dollars. Domestic availability of duced into this country and then became a zoonotic risk. farmed fi sh is also a way of increasing protein availability

Figure 3. Adult trematodes isolated from Vietnamese persons. A) Haplorchis pumilio. B) H. taichui. C) H. yokogawai. D) Stellantchasmus falcatus. (Semichon acetocarmine stained, magniﬁ cation ×120.)

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1831 RESEARCH

Helminthology Department, Faculty of Tropical Medicine, Ma- hidol University, for their assistance with many aspects of this study. We also thank the staff at the Fishborne Zoonotic Parasites in Vietnam project secretariat, especially Jesper Clausen, Jacob Fjalland, and Bui Thanh, for indispensable assistance and coop- eration. This study was supported by the Danish International Devel- opment Assistance, the Research Council for Development Re- search, and the Fisheries Sector Program Support in Vietnam, as part of the research capacity building project, Fishborne Zoonotic Parasites in Vietnam (www.ﬁ bozopa.rial.org) project no. 91140. Dr Dung is a research scientist in the Department of Parasi- tology, National Institute of Malariology, Parasitology and Ento- mology, Hanoi. His research interests include detection and control of zoonotic parasite diseases. Figure 4. Multiple ﬁ shborne trematode infections in humans, Nam Dinh Province, Vietnam, April 2005. References

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